Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin

In the present study, we developed an antitumor immunoconjugate that appears to be promising as a novel curative antitumor agent against a variety of human solid tumors. We generated a new antihuman endoglin (EDG) monoclonal antibody (mAb) K4-2C10 (or termed SN6f) that cross-reacts with mouse endothelial cells. Such cross-reactive anti-EDG mAbs have not been reported previously. This mAb was used to target tumor-associated vasculature in SCID mice inoculated with human tumors. No anti-EDG mAb or its immunoconjugates have previously been successfully used for targeting vasculature in vivo. In this study, MCF-7 human breast cancer cells were inoculated s.c. into SCID mice. K4-2C10 did not react with the MCF-7 cells but showed a weak reactivity with mouse endothelial cells. The mAb reacted with the proliferating endothelial cells more strongly than with the quiescent endothelial cells. The mAb exhibited much stronger reactivity (>10-fold) with human endothelial cells than with mouse endothelial cells and reacted strongly with vascular endothelium of tumor-associated blood vessels in a variety of human malignant tissues. Conjugates of K4-2C10 with ricin A chain (RA) and deglycosylated ricin A chain (dgRA) showed a weak but specific cytotoxic activity against murine endothelial cells in vitro; the 50% inhibitory dose of the RA and dgRA conjugates was 54 nm and 29 nm, respectively. Remarkable antitumor efficacy was observed when a small amount (a total of 60 microgram corresponding to 24% of the LD50 dose) of the dgRA conjugate was administered i.v. into SCID mice that had been inoculated s.c. with MCF-7. Unconjugated mAb K4-2C10 was not significantly effective in the inhibition of the tumor growth. The immunotoxin (IT) completely inhibited growth of the tumor in all of the treated mice (n = 8). Furthermore, similar antitumor efficacy was observed when the IT was administered i.v. into the tumor-inoculated SCID mice that had been pretreated with unconjugated K4-2C10 to block the potentially available weak binding sites of normal tissues. The strong therapeutic effects of the IT were reproduced in another set of therapeutic experiments. No significant side effects were observed in the mice. The differences in the tumor growth between the control group and the IT-treated groups were statistically significant. The IT showed antiangiogenic activity in the dorsal air sac method. The results indicate that K4-2C10 IT effectively treated the tumor-bearing mice by selectively inhibiting the tumor-associated blood vessels and by disrupting tumor-associated angiogenesis. The strong antitumor efficacy of the K4-2C10 IT is remarkable in view of the fact that K4-2C10 and its IT showed only a weak reactivity with mouse endothelial cells, and a relatively small amount of the IT was administered i.v. to treat s.c. tumors. We anticipate that the K4-2C10 IT will show much stronger antitumor efficacy and antiangiogenic activity in patients with solid tumors and other angiogenesis-associated diseases. The present results demonstrate for the first time that an anti-EDG mAb or its immunoconjugate can effectively target tumor-associated vasculature in vivo.     

Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice

Parvovirus minute virus of mice strain i (MVMi) infects committed granulocyte-macrophage CFU and erythroid burst-forming unit (CFU-GM and BFU-E, respectively) and pluripotent (CFU-S) mouse hematopoietic progenitors in vitro. To study the effects of MVMi infection on mouse hemopoiesis in the absence of a specific immune response, adult SCID mice were inoculated by the natural intranasal route of infection and monitored for hematopoietic and viral multiplication parameters. Infected animals developed a very severe viral-dose-dependent leukopenia by 30 days postinfection (d.p.i.) that led to death within 100 days, even though the number of circulating platelets and erythrocytes remained unaltered throughout the disease. In the bone marrow of every lethally inoculated mouse, a deep suppression of CFU-GM and BFU-E clonogenic progenitors occurring during the 20- to 35-d.p.i. interval corresponded with the maximal MVMi production, as determined by the accumulation of virus DNA replicative intermediates and the yield of infectious virus. Viral productive infection was limited to a small subset of primitive cells expressing the major replicative viral antigen (NS-1 protein), the numbers of which declined with the disease. However, the infection induced a sharp and lasting unbalance of the marrow hemopoiesis, denoted by a marked depletion of granulomacrophagic cells (GR-1(+) and MAC-1(+)) concomitant with a twofold absolute increase in erythroid cells (TER-119(+)). A stimulated definitive erythropoiesis in the infected mice was further evidenced by a 12-fold increase per femur of recognizable proerythroblasts, a quantitative apoptosis confined to uninfected TER-119(+) cells, as well as by a 4-fold elevation in the number of circulating reticulocytes. Therefore, MVMi targets and suppresses primitive hemopoietic progenitors leading to a very severe leukopenia, but compensatory mechanisms are mounted specifically by the erythroid lineage that maintain an effective erythropoiesis. The results show that infection of SCID mice with the parvovirus MVMi causes a novel dysregulation of murine hemopoiesis in vivo.     

Growth of human T-cell lineage acute leukemia in severe combined immunodeficiency (SCID) mice and non-obese diabetic SCID mice

Primary leukemic cells from patients with acute lymphoblastic leukemia (ALL) can be injected intravenously into mice with severe combined immunodeficiency (SCID) to create a model of human leukemia. Leukemic cells disseminate to murine tissues in a clinicopathologic pattern similar to that seen in humans. Thus far, reports of engraftment of lymphoid leukemia in SCID mice have mainly been from patients with B-cell lineage ALL, for which engraftment occurs more frequently with cells from high-risk patients. There are few data on the engraftment of T-cell lineage ALL in SCID mice. Leukemic cells from 19 patients (16 adult and three pediatric) with T-cell lineage ALL were injected into SCID mice, with overt engraftment of 12 cases (63%). Engraftment of leukemia in SCID mice was associated with earlier death due to leukemia of the patient donors (P < .01, log-rank test). The recently developed non-obese diabetic (NOD)/SCID mouse may expand the uses of the SCID model. Cells from the seven patients with T-cell lineage ALL that failed to cause leukemia in SCID mice were injected into NOD/SCID mice. Overt leukemia engraftment was observed in all seven cases. Thus, growth of human T-cell lineage ALL cells in SCID mice was associated with a high-risk patient group. However, this association was not observed when NOD/SCID mice were used, suggesting that this model would no longer predict patients likely to die early of leukemia, but may provide a more realistic system for studying the biology and treatment of the disease.     

A new method for evaluating the rheological properties of the erythrocytes in surgical patients with endogenous intoxication

An examination of 30 patients against the background of using both the operative and conservative methods of treatment was performed in order to study the rheological properties of blood and selection of the most informative tests for diagnosis of microcirculatory disturbances in diseases of the gastrointestinal tract and gastroduodenal area. The authors propose methods of investigation of deformability and viscosity of erythrocytes in patients with the surgical diseases in question. The methods are simple, quite handy for any surgical hospital and polyclinic laboratories. In addition to being used in investigations of the aggregative ability of erythrocytes and viscosity of the whole blood, these methods can be of use in express diagnosis of rheological disorders. The endogenous intoxication in patients with the diseases in question was found to be accompanied by deep rheological disturbances. The deformability of erythrocytes and their viscosity index which can pass ahead of the shifts in other parameters characterizing the hemocoagulative and rheological properties of blood are changed. A conclusion is drawn that the parameters of the erythrocyte deformability and viscosity can be an additional criteria of the adequacy of therapy used for patients with the diseases in question.     

The use of an anti-CD3 immunotoxin to prevent the development of lymphoproliferative disease in SCID/PBL mice

Severe combined immunodeficient mice (SCID) reconstituted with normal PBLs (SCID/PBL) from Epstein-Barr virus-positive (EBV+) human donors often develop fatal human B lymphomas which resemble the EBV-induced lymphoproliferative disease (LPD) observed in immunosuppressed individuals. This phenomenon appears to be T cell dependent. In this study we used an immunotoxin (IT) prepared by conjugating the monoclonal anti-CD3 antibody, 64.1, to deglycosylated ricin A chain (dgRTA) to prevent LPD in SCID/PBL mice. We show that the incidence of LPD is greatly reduced by either a combination of in vitro treatment of PBLs followed by one in vivo treatment of the xenografted mice with 64.1-dgRTA immunotoxin or by repeated treatments in vivo with the immunotoxin. In contrast, in vitro treatment alone or in vivo treatment with only one injection of 64.1-dgRTA were less effective. As expected, this IT did not have any non-specific cytotoxic effects on already established EBV+ tumors from SCID/PBL mice. The use of this IT, therefore, represents a simple method to avoid LPD when injecting blood-containing tissues into SCID mice.     

A rapid method for determining the particle-size distribution of micropowders

Conclusions Use of a flow-type laser analyzer in an investigation of the particle size distribution of a powder markedly accelerates analysis and enables the latter to be performed automatically, with the recording of numerical characteristics and forms of distribution functions for particles ranging in size from a few hundreds of angstrom units to some tens of micrometers. It is shown that the distribution functions of the particles of titanium carbide powders produced by comminution in a ball mill and subsequent liquid centrifugal classification into 3–5-, 2–3-, and 1–3-m fractions obey the logarithmic normal law. The numerical characteristics of these distributions are given.Translated from Poroshkovaya Metallurgiya, No. 11(263), pp. 10–14, November, 1984.     

A new device for the rapid measurement of impaired motor function in mice

Measurement of the ability of mice to balance on a rotating rod or cone is often used as a measure of impaired motor function. In most of these procedures the mice must be trained prior to the test. In the new screen test described in this paper, untrained mice are used in a 60 sec test which measures the ability of mice to either climb to the top of or cling to the bottom of a horizontal screen. The ED50 values obtained for failure to reach the top of the horizontal screen are similar to those obtained with the rotarod; the values for falling from the screen are somewhat higher. With both of the horizontal screen measures there were fewer control failures than in the rotarod procedure.     

A rapid method for disrupting genes in the Escherichia coli genome

The entire genomic sequence of Escherichia coli has recently been completed. To gain insight into the function of the vast array of yet uncharacterized open reading frames (ORFs), a variety of new genetic tools will be required. Here we examined a genetic system, using an integration plasmid vector (named pINT007), for rapid construction of disruption mutants of any ORF in E. coli. It was found that the vector allows us to rapidly construct a disruption mutant for any gene on the chromosome as a cointegrate, furthermore, resolution of the resulting cointegrate can be surely accomplished by using a pair of the bla (ampicillin resistant) genes on the vector as a positive-selection marker.     

A rapid method for the regional dissection of the rat brain

A method is described for the rapid dissection of seventeen areas of the rat brain. Regions from fresh unfrozen brain tissue are dissected from coronal brain slices obtained with use of a cutting block. This method is applicable to pharmacological and behavioral studies which require the dissection of numerous brains during short time intervals.     

A filter immunobinding technique for the rapid detection and simultaneous identification of avian and bovine mycoplasmas

A filter immunobinding (FIB) method was developed for the detection and identification of mycoplasmas. Type strains of a total of 18 avian and bovine mycoplasma species propagated in broth media were diluted and immobilized on a nitrocellulose membrane as antigens for investigating the specificity with rabbit hyperimmune sera. Non-specific FIB reactions were easily eliminated by the procedure of absorbing rabbit hyperimmune sera in the broth. Absorbed rabbit hyperimmune sera exhibited clear species-specificity with mycoplasma antigens by the FIB. These specific reactions always agreed with the results of identification by tests of biochemical properties and growth inhibition for the isolates of M. bovirhinis, M. bovis, M. columbinum, M. columborale, M. gallisepticum and M. synoviae. Some bovine mycoplasma species, which were impossible to identify by growth inhibition test, because of their strong production of film and spots on the agar, specifically reacted with absorbed rabbit hyperimmune sera against M. bovis in the FIB. The detection limit of mycoplasmas by this method was about 10(4)-10(5) colony-forming units/ml, which is lower than that of colony determination on agar. The FIB seems to be a useful technique for rapid detection and simultaneous identification of mycoplasmas.     

A rapid method for regulating xenobiotics in the atmosphere for chemical accident situations

PURPOSE: The purpose of this study was to survey dental hygiene graduates to determine utilization in the private practice setting of expanded functions taught in school. METHOD: A questionnaire was sent to 90 dental hygiene graduates from the classes of 1990-1994, of Indiana University-Purdue University, Fort Wayne. Analysis was done on utilization of specific expanded functions taught in school. Frequency tabulations of procedures performed and adequacy of preparation were done using the SAS software. The results were graphically represented in tables. RESULTS: Equal response from each class resulted in the total response rate of 74%. The expanded functions most frequently performed by dental hygienists were placing sealants followed by making cast impressions. Placing temporary and amalgam restorations were rarely performed. The majority of graduates felt adequately trained to perform these skills. CONCLUSION: Skills being required and taught in school are not being delegated in private practice. This survey shows the need for curriculum revisions and discrepancies between educational requirements and utilization.     

Study of Dengue virus infection in SCID mice engrafted with human K562 cells

Here we report that severe combined immunodeficient (SCID) mice engrafted with human K562 cells (K562-SCID mice) can be used as an animal model to study dengue virus (DEN) infection. After intratumor injection into K562 cell masses of PL046, a Taiwanese DEN-2 human isolate, the K562-SCID mice showed neurological signs of paralysis and died at approximately 2 weeks postinfection. In addition to being detected in the tumor masses, high virus titers were detected in the peripheral blood and the brain tissues, indicating that DEN had replicated in the infected K562-SCID mice. In contrast, the SCID mice were resistant to DEN infection and the mock-infected K562-SCID mice survived for over 3 months. These data illustrate that DEN infection contributed directly to the deaths of the infected K562-SCID mice. Other serotypes of DEN were also used to infect the K562-SCID mice, and the mortality rates of the infected mice varied with different challenge strains, suggesting the existence of diverse degrees of virulence among DENs. To determine whether a neutralizing antibody against DEN in vitro was also protective in vivo, the K562-SCID mice were challenged with DEN-2 and received antibody administration at the same time or 1 day earlier. Our results revealed that the antibody-treated mice exhibited a reduction in mortality and a delay of paralysis onset after DEN infection. In contrast to K562-SCID, the persistently DEN-infected K562 cells generated in vitro invariably failed to be implanted in the mice. It seems that in the early stage of implantation, a gamma interferon activated, nitric oxide-mediated anti-DEN effect might play a role in the innate immunity against DEN-infected cells. The system described herein offers an opportunity to explore DEN replication in vivo and to test various antiviral protocols in infected hosts.     

Effects of immunopharmacological compounds in the Lyme arthritis model using normal and SCID mice

The murine Lyme borreliosis causes a special type of arthritis whose development appears to be controlled by a functioning immune system. Immunocompetent C3H and severe combined immunodeficient (SCID) mice were infected with Borrelia burgdorferi (strain SH-2-82) to induce experimental Lyme disease. Expression of clinical symptoms was mild to very moderate in the C3H but more rapidly developing and severe in the SCID mouse. Various pharmacological compounds, such as anti-inflammatory and immunosuppressive drugs, monoclonal antibodies and other miscellaneous agents, were investigated for profiling their effects in this model in both mouse strains. Several disease parameters were assessed, in particular paw swelling. The use of these various compounds provided further evidence that experimental borreliosis in mice represents a special type of arthritis which has no autoimmune basis and which requires productive infection with Borrelia burgdorferi. In addition, when comparing these results with those obtained in other mainly immune driven arthritis models commonly used in inflammation research, it is concluded that this arthritis model is not suitable for the therapeutic assessment of antiinflammatory agents.     

Growth and metastasis of two canine mast cell tumors in SCID mice

Intermediately differentiated mast cell tumors in two dogs were subcutaneously xenotransplanted into severe combined immunodeficiency (SCID) mice. Both tumors primarily grew and were serially transplantable in SCID mice. The histological features of the xenografts were similar to those of original tumors in dogs. Both of these subcutaneous tumors were judged as connective tissue mast cells by toluidine blue stain. One of the two xenografts metastasized to the tracheobronchial lymph nodes, omentum, mesentery, subpleural region and retroperitoneum of the SCID mouse. These canine mast cell tumor xenografts in SCID mice may be valuable tools for investigating the growth and metastatic behaviors of the tumor.